Advancing spatial oncology research with open-panel multiplex IHC/IF using a novel heat-free antibody stripping reagent

Purpose: Spatially resolved multiplex immunohistochemistry (IHC) and immunofluorescence (IF) assays are essential for profiling the tumor microenvironment and identifying biomarkers that predict response to cancer immunotherapies, yet many platforms rely on proprietary closed antibody panels and harsh, heat-based stripping that damage FFPE tissue and reduce antigenicity, limiting target choice and rapid adoption of new markers. We developed a heat-free stripping reagent that removes primary and secondary antibodies and other non-covalently bound detection reagents between staining cycles while preserving tissue architecture and antigen integrity. This abstract summarizes its performance in multiplex IHC, IF, and tyramide-based workflows and its compatibility with common autostainers. Experimental Procedures: FFPE tumor and normal tissues were stained using sequential multiplex IHC, IF and TSA workflows in both manual and automated staining, with panels targeting immune, stromal, and tumor markers. After each staining round, slides were treated with the heat-free reagent before the next cycle. Residual signal was assessed by imaging previously used channels after stripping and by chase experiments to detect previously applied antibodies. Antigen preservation was evaluated by comparing staining intensity and distribution for the same targets across early and late cycles, and morphology by H&E. Summary of New Data: The stripping reagent consistently removed bound antibodies and associated detection reagents, with no detectable residual signal in previously used channels. Across multiple sequential rounds, key tumor and immune markers maintained expected expression patterns and subcellular localization, with no appreciable loss of antigen detectability. Tissue morphology remained intact after repeated stain-strip cycles. The reagent performed robustly in chromogenic IHC, IF, and TSA multiplex workflows, enabling multi-marker panels on a single section without cross-reactivity or signal carryover. Protocols were easily adapted to autostainers, integrating into translational and biomarker pipelines. Conclusions: This heat-free stripping reagent enables robust multiplex IHC, IF, and TSA staining on FFPE tissue while preserving morphology and antigenicity across multiple sequential cycles and is readily adaptable to autostainers. It simplifies adoption of multiplex panels in oncology research, conserves clinical specimens, and enables deeper characterization of the tumor immune microenvironment and biomarkers for precision oncology. Unlike closed multiplex platforms, it is compatible with investigator-selected antibodies and detection reagents, supporting flexible panels that keep pace with evolving oncology targets.